The fundamental goal of the research program continues to be the elucidation of molecular mechanisms involved in the coordination and control of DNA replication in cultured human cells. We have reported the successful establishment and preliminary characterization of 16 stable murine monoclones that produce homogeneous antibodies against human DNA polymerase-alpha. Three of the antibodies possess potent anti-polymerase-alpha neutralizing activities; none of the 16 antibodies detectably interacts with DNA polymerases-beta or -gamma. A major effort to date has been to use several of these antibodies for development of rapid, highly efficient immunopurification protocols by which it is possible to prepare DNA polymerase-alpha fractions of extraordinarily high specific activity from moderate amounts of cultured cells in only 3 days. These immunoaffinitypurified fractions contain an interesting set of polypeptides, including a family of large polypeptides that range between 200,000 and 130,000 daltons. We have obtained evidence that the epitopes recognized by all of our monoclonal antibodies map within this family of large polypeptides and that at least some of the peptides in this family possess DNA polymerase-alpha catalytic activity. We have identified and characterized in detail a DNA primase activity that is present in the immunoaffinity-purified polymerase-alpha fractions and have developed an entirely novel model of primase activity that involves the tightly synchronized coupling of that activity with polymerase-alpha. Several of the monoclonal antibodies have been used in a variety of collaborative studies with significant results: (1) an initial cytochemical study demonstrated in three separate lines of cultured human cells the exclusively intranuclear locations of DNA polymerasealpha antigens, and (2) a related study demonstrated that none of the 16 monoclonal antibodies is capable of interacting with glucocorticoid receptors prepared from KB cells. (3) We have demonstrated the capacity of several of the monoclonal antibodies to inhibit nuclear DNA replication as measured in a commonly utilized permeabilized cell system. (4) Studies to date indicate that the neutralizing antibodies are highly interspecifically cross-reactive, whereas at least several of the non-neutralizing, binding activities seem to recognize epitopes that are limited exclusively to KB cell (or human) DNA polymerase-alpha species. These preliminary observations have also led to a final collaborative study to identify the human chromosomal locus of polymerasealpha by use of interspecific (human-mouse) hybrids.